The Interferon Consensus Sequence Binding Protein (Icsbp or Irf8) is a transcription factor that functions as a leukemia-suppressor. We identified an Icsbp-target-gene set that is enriched for genes that control Fas and/or catenin activity. This is of interest, because decreased expression of Icsbp, Fas-resistance and increased catenin activity are associated with poor prognosis and disease progression in chronic myeloid leukemia (CML). Insensitivity of leukemia stem cells (LSC) to Fas-induced apoptosis is associated with development of drug resistance in CML, but does not correlate with decreased expression of Fas or FasL. Increased catenin activity precedes blast crisis (BC) in CML, but does not correlate with Wnt expression or CTNNB1 transcription. We determined that Icsbp represses the gene encoding Fap1; a Fas inhibitory protein. We found Icsbp/Fap1-dependent Fas-resistance in Bcr-abl+ cells. Fap1 also interacts with Apc, and we found Icsbp/Fap1/Gsk3b-dependent catenin stabilization in these cells. We identified GAS2 as another Icsbp- target-gene. Gas2 inhibits calpain; a protease with substrates that include catenin, Stat5 and Xiap. We found a Gas2/calpain-dependent increase in catenin in Bcr-abl+ or Icsbp-/- murine bone marrow cells. Stat5 and Xiap are also increased in Icsbp-/- cells in a Gas2/calpain-dependent manner. Xiap contribute to Fas- resistance, and we found that Stat5 represses the IRF8 promoter in a Gas2/calpain-dependent manner. Although tyrosine kinase inhibitors (TKI) induce remission in the majority of CML patients, an LSC sub- population persists during treatment, preventing cure with TKIs. The hypothesis of these studies is that Fap1, calpain and related pathways are rational therapeutic targets to abolish the persistent CML-LSC population; preventing emergence of overt drug-resistance and/or BC. This hypothesis will be pursued by 3 Aims; AIM 1: Define roles of calpain and Fap1 in development of TKI resistance in CML. The roles of Fap1 and calpain in Fas resistance will be studied in vitro using primary murine bone marrow cells and human CML bone marrow samples. The impact of targeting Fap1 or calpain on development of TKI resistance will be investigated in vivo in a murine CML bone marrow transplant model. AIM 2: Determine if cooperation between calpain and Fap1 facilitates BC in CML. Regulation of catenin activity by Fap1 and calpain in will be studied in vitro using murine bone marrow cells and human CML samples. The impact of targeting Fap1 or calpain on BC will be studied in vivo in a murine CML model. AIM 3: Investigate the impact of calpain activation on Icsbp-expression in CML. Regulation of Icsbp expression by Bcr-abl-dependent Stat5 activation will be studied in myeloid cell lines and primary murine bone marrow cells. Targeting calpain to increase Icsbp will be investigated in vivo in murine CML models. The goal of these studies is to identify molecular mechanisms that lead to LSC persistence, and therefore TKI resistance and/or BC in CML. Targeting these mechanisms might cure CML by abolishing the LSC.